ATX3 Antibody

What is ATX3/ATXN3 and what cellular functions does it perform?

Ataxin-3 (ATX3/ATXN3) is a deubiquitinating enzyme that plays critical roles in multiple cellular processes including protein homeostasis maintenance, transcription regulation, cytoskeleton organization, myogenesis, and the degradation of misfolded chaperone substrates . The protein functions primarily by binding long polyubiquitin chains and trimming them, with significantly reduced activity against chains of 4 or fewer ubiquitins . Mutations in ATXN3, particularly CAG repeat expansions, are associated with Spinocerebellar ataxia-3 (SCA3), also known as Machado Joseph Disease, an incurable neurodegenerative disorder . The wild-type allele typically contains approximately 24 CAG repeats, while the mutant allele in SCA3 patients often contains significantly more repeats, such as the 74 repeats observed in certain patient-derived fibroblast cell lines . This expanded CAG repeat results in a polyglutamine-expanded ataxin-3 protein that exhibits altered aggregation properties and cellular toxicity.

What are the primary applications for ATX3/ATXN3 antibodies in laboratory research?

ATX3/ATXN3 antibodies have been validated for multiple experimental applications, with different antibodies showing variation in their optimal applications. Based on published research, the primary applications include:

For robust experimental design, researchers should consider the extensively documented reactivity of available antibodies with human, mouse, and rat samples, with some antibodies also showing validated reactivity with pig and C. elegans models .

What is the expected molecular weight of ATX3/ATXN3 in Western blot applications?

When performing Western blot analysis using ATX3/ATXN3 antibodies, researchers should expect to observe bands at the following molecular weights:

• Calculated molecular weight: 43 kDa (based on 370 amino acids)

• Observed molecular weight range: 35-42 kDa

The variation between calculated and observed molecular weights may result from post-translational modifications, protein processing, or the specific conditions of the electrophoresis system used. Additionally, researchers studying both wild-type and mutant forms should expect to see differences in band migration patterns due to the expanded polyglutamine tract in the mutant form. The wild-type form (typically 24 CAG repeats) will migrate faster than the mutant form (e.g., 74 CAG repeats in SCA3 patients) .

What are the optimal conditions for detecting ATX3/ATXN3 via Western blot analysis?

For optimal Western blot detection of ATX3/ATXN3, consider the following methodological parameters:

It is essential to optimize these conditions for your specific experimental system. When studying both wild-type and mutant forms, use gradient gels (e.g., 4-12%) to achieve better separation between the differentially sized proteins. Additionally, include positive controls from validated cell lines to ensure the detection system is functioning properly. For challenging applications or when signal-to-noise ratio is suboptimal, consider titrating the antibody concentration and extending primary antibody incubation time (overnight at 4°C).

How can researchers effectively monitor ATX3/ATXN3 aggregation in experimental models?

Monitoring ATX3/ATXN3 aggregation, particularly for studies investigating polyQ-expanded variants, requires specialized techniques. A robust approach combines thioflavin-T (ThT) fluorescence assays with transmission electron microscopy (TEM):

• Miniaturized ThT Fluorescence Assay:

  • This approach allows for real-time monitoring of amyloid fibril formation

  • Can be performed in 96-well plate format for high-throughput screening

  • Enables quantitative assessment of aggregation kinetics

• TEM Visualization:

  • Provides direct visualization of aggregate morphology

  • Confirms the formation of amyloid fibrils

  • Allows characterization of structural features of aggregates

When establishing ATX3/ATXN3 aggregation assays, researchers should systematically evaluate the impact of various experimental conditions including:

• Ionic strength variations

• pH range (typically 4.0-8.0)

• Presence of detergents (SDS, Triton X-100)

• Molecular crowding agents (PEG, dextran)

• Protein concentration

For studies focusing on inhibitor screening, this combined approach provides a platform to evaluate potential therapeutic compounds or peptides, such as polyQ binding peptide 1 (QBP1) and linear ubiquitin chains, which have been reported to modulate ataxin-3 aggregation .

What strategies can be employed for allele-selective inhibition of mutant ATX3/ATXN3 expression?

For researchers investigating therapeutic approaches to SCA3/MJD, allele-selective inhibition strategies targeting the mutant ATX3/ATXN3 while preserving wild-type expression are of paramount importance. Several nucleic acid-based approaches have shown promise:

• Peptide Nucleic Acids (PNAs):

  • PNAs of various lengths (7-19 bases) conjugated to cationic peptides show selective inhibition of mutant ATX3/ATXN3

  • IC₅₀ values ranging from 0.5-0.6 μM with 2.4-3.6 fold selectivity for mutant versus wild-type allele

  • PNA length affects potency and selectivity, with 7-base PNAs showing comparable efficacy to longer constructs

• Modified PNA-Peptide Conjugates:

  • The position of the cationic peptide (N vs. C terminus) affects selectivity

  • C-terminal lysine conjugates (IC₅₀ = 1.7 μM) vs. N-terminal lysine conjugates (IC₅₀ = 3.5 μM)

  • Arginine-containing peptides show enhanced potency (C-terminal arginine IC₅₀ = 0.7 μM)

• Mismatched Duplex RNAs:

  • Introduction of specific mismatches in anti-CAG duplex RNAs enhances selectivity

  • Position 6 in the seed sequence is critical for target recognition

  • Multiple mismatches (2-4) may increase selectivity

These approaches exploit structural differences between wild-type and expanded CAG repeat regions in ATX3/ATXN3 mRNA. The expanded CAG repeats in mutant transcripts form hairpin structures that differ from those in wild-type, allowing selective targeting. When designing such inhibitors, researchers must carefully optimize oligomer length, chemistry, conjugation strategy, and mismatch position to achieve maximum selectivity.

What are the optimal conditions for immunohistochemical detection of ATX3/ATXN3 in tissue samples?

For successful immunohistochemical detection of ATX3/ATXN3 in tissue sections, researchers should consider the following protocol optimizations:

When working with neurological tissues, particularly in the context of SCA3/MJD research, consider these additional methodological refinements:

• Optimize fixation time to preserve tissue architecture while ensuring adequate antibody penetration

• For double-labeling experiments, use fluorescent secondary antibodies with distinct emission spectra

• Include wild-type and SCA3 patient-derived tissues for comparative analysis when available

• Consider thinner sections (5-7 μm) for improved resolution of subcellular localization

• For aggregation studies, compare results with thioflavin staining in adjacent sections

These protocol adaptations are particularly important when studying the different aggregation patterns between wild-type ATX3 (typically 13Q) and expanded polyQ ATX3 (e.g., 77Q) in neuronal tissues .

How can ATX3/ATXN3 antibodies be utilized to study protein-protein interactions?

ATX3/ATXN3 participates in numerous protein-protein interactions that are crucial for its normal function and disease pathogenesis. To investigate these interactions, researchers can employ several antibody-based approaches:

• Co-Immunoprecipitation (Co-IP):

  • ATX3/ATXN3 antibodies have been validated for Co-IP applications in multiple studies

  • Recommended antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

  • Suitable for identifying novel interaction partners or confirming predicted interactions

• Proximity Ligation Assay (PLA):

  • Provides in situ visualization of protein interactions with single-molecule sensitivity

  • Requires specific antibodies raised in different host species

  • Particularly useful for detecting transient or weak interactions

• FRET/BRET Analysis with Antibody Validation:

  • Fluorescence/Bioluminescence Resonance Energy Transfer techniques

  • ATX3/ATXN3 antibodies serve as validation tools for tagged constructs

  • Enables real-time monitoring of interactions in living cells

When designing interaction studies, researchers should consider:

• The impact of polyQ expansion on interaction profiles

• The role of ubiquitin-binding domains in mediating interactions

• Potential conformational changes affecting antibody epitope accessibility

• The importance of STUB1/CHIP interactions in degradation of misfolded chaperone substrates

What considerations are important when using ATX3/ATXN3 antibodies in neurodegeneration research?

When applying ATX3/ATXN3 antibodies in neurodegeneration research, particularly for SCA3/MJD studies, researchers should address several critical considerations:

• Epitope Selection and Accessibility:

  • Antibodies targeting regions outside the polyQ tract can detect both wild-type and mutant forms

  • Epitopes within or adjacent to the polyQ region may have differential accessibility in aggregated states

  • Consider using multiple antibodies targeting different epitopes for comprehensive analysis

• Aggregate Detection Specificity:

  • Confirm antibody specificity using knockout/knockdown controls

  • Validate reactivity in disease-relevant tissues (human, mouse, rat)

  • Compare results with aggregate-specific dyes (ThT, Congo Red) in parallel experiments

• Model System Selection:

  • Patient-derived fibroblasts (e.g., GM06151 with 24/74 CAG repeats) provide relevant cellular models

  • Consider species differences when working with animal models

  • Neuron-specific effects may not be fully recapitulated in non-neuronal cells

• Temporal Considerations:

  • ATX3/ATXN3 aggregation and toxicity develop progressively

  • Design time-course experiments to capture early events prior to overt pathology

  • Consider age-dependent changes in protein expression and localization

For therapeutic development studies, researchers should integrate antibody-based detection methods with functional assays to correlate molecular changes with phenotypic outcomes. The combined approach of using antibodies for protein detection alongside aggregation monitoring techniques provides a more comprehensive understanding of disease mechanisms .

How do experimental conditions affect ATX3/ATXN3 aggregation assays?

The aggregation of ATX3/ATXN3, particularly the expanded polyQ variants, is highly sensitive to experimental conditions. Researchers should systematically evaluate these parameters when establishing robust aggregation assays:

When developing ATX3/ATXN3 aggregation assays, a miniaturized ThT fluorescence approach combined with TEM visualization provides complementary quantitative and qualitative data . This platform can be used to:

• Evaluate aggregation kinetics through real-time fluorescence monitoring

• Characterize aggregate morphology via electron microscopy

• Screen potential aggregation modulators including:

  • PolyQ binding peptide 1 (QBP1)

  • Linear ubiquitin chains

  • Small molecule inhibitors

  • Chaperone proteins

For meaningful results, researchers should include both wild-type (Atx3 13Q) and polyQ-expanded (Atx3 77Q) proteins in parallel experiments to distinguish polyQ-dependent from polyQ-independent aggregation mechanisms .

What validation controls are essential when using ATX3/ATXN3 antibodies?

Rigorous validation is crucial for ensuring reliable results with ATX3/ATXN3 antibodies. Researchers should implement the following controls:

• Specificity Controls:

  • Knockout/knockdown samples (validated in 6+ publications)

  • Competing peptide blocking

  • Multiple antibodies targeting different epitopes

  • Western blot showing single band at expected molecular weight (35-42 kDa)

• Application-Specific Controls:

  • WB: Positive control lysates (Neuro-2a cells, HEK-293 cells, mouse/rat brain tissue)

  • IHC: Known positive tissues with appropriate antigen retrieval methods

  • IF: Secondary antibody-only controls

  • IP: Non-immune IgG controls

• Disease Model Validation:

  • Compare wild-type and mutant ATX3/ATXN3 detection

  • Patient-derived samples versus controls

  • Age-matched samples for developmental/degenerative studies

When publishing results, researchers should provide comprehensive details of validation procedures and include representative images of control experiments. This practice enhances reproducibility and confidence in the findings. For knockdown/knockout validation, quantitative assessment of signal reduction (typically >80% reduction expected) should be reported alongside representative images.

How should researchers address technical challenges in detecting aggregated forms of ATX3/ATXN3?

Detection of aggregated ATX3/ATXN3 presents unique technical challenges that researchers must address through methodological adaptations:

• Sample Preparation Considerations:

  • Aggregates may be resistant to standard lysis buffers

  • Consider sequential extraction protocols (detergent-soluble → detergent-insoluble → formic acid-soluble fractions)

  • Avoid excessive sonication which may disrupt aggregate structure

• Electrophoresis Modifications:

  • Use gradient gels (4-20%) to resolve monomeric and aggregated species

  • Include stacking gel retention analysis to detect high-molecular-weight aggregates

  • Consider native PAGE for preservation of aggregate structure

• Microscopy Optimizations:

  • Combine immunofluorescence with aggregate-specific dyes

  • Use super-resolution techniques (STED, STORM) for detailed aggregate morphology

  • Consider FRAP (Fluorescence Recovery After Photobleaching) to assess aggregate dynamics

• Antibody Selection Strategy:

  • Test antibodies against different epitopes (N-terminal, C-terminal, polyQ region)

  • Some epitopes may become masked in aggregated states

  • Consider conformational antibodies specifically recognizing misfolded species

For robust aggregation analysis in the context of SCA3/MJD research, the combination of ThT fluorescence assays with TEM visualization provides complementary insights into both aggregation kinetics and morphology . This approach can be supplemented with filter trap assays and dynamic light scattering for comprehensive characterization of aggregation intermediates and mature fibrils.

What emerging technologies might enhance ATX3/ATXN3 research?

Several cutting-edge technologies show promise for advancing ATX3/ATXN3 research:

• CRISPR-Based Approaches:

  • Base editing for precise correction of CAG expansions

  • CRISPRi for selective silencing of mutant alleles

  • CRISPR activation systems to upregulate compensatory pathways

• Advanced Imaging Technologies:

  • Cryo-electron microscopy for atomic-resolution structures of ATX3/ATXN3 aggregates

  • Lattice light-sheet microscopy for long-term live-cell imaging of aggregate formation

  • Expansion microscopy for enhanced visualization of subcellular aggregate distribution

• Single-Cell Technologies:

  • Single-cell proteomics to detect cell-to-cell variation in ATX3/ATXN3 expression

  • Spatial transcriptomics to map regional variation in neuronal vulnerability

  • Time-resolved single-cell analysis of aggregation events

• Therapeutic Approaches:

  • Allele-selective oligonucleotides with enhanced specificity for mutant transcripts

  • PNA-peptide conjugates optimized for blood-brain barrier penetration

  • Mismatched duplex RNAs with improved selectivity profiles

The development of selective inhibition strategies targeting mutant ATX3/ATXN3 while preserving wild-type protein function represents a particularly promising research direction. Recent advances with PNAs of varied lengths (7-19 bases) achieving IC₅₀ values of 0.5-0.6 μM and selectivities of 2.4-3.6 fold for the mutant allele demonstrate the feasibility of this approach . Further optimization of oligomer design, particularly through the strategic introduction of mismatches, may enhance selectivity and therapeutic potential.

How might ATX3/ATXN3 antibodies contribute to therapeutic development for SCA3/MJD?

ATX3/ATXN3 antibodies play crucial roles in therapeutic development for SCA3/MJD beyond their utility as research tools:

• Target Validation and Mechanism Studies:

  • Confirm target engagement of therapeutic candidates

  • Characterize mechanisms of action for aggregation inhibitors

  • Monitor changes in ATX3/ATXN3 levels and distribution following treatment

• Biomarker Development:

  • Quantify soluble vs. aggregated ATX3/ATXN3 in accessible biofluids

  • Monitor disease progression through longitudinal sampling

  • Stratify patient populations for clinical trials

• Therapeutic Antibody Development:

  • Intrabodies targeting specific ATX3/ATXN3 conformations

  • Antibody-drug conjugates for selective targeting of cells with aggregates

  • Single-domain antibodies optimized for intracellular delivery

• Supporting Gene Therapy Approaches:

  • Validate efficacy of gene silencing approaches

  • Monitor selectivity of allele-specific therapies targeting expanded CAG repeats

  • Assess biodistribution and durability of genetic interventions

The selective inhibition of mutant ATX3/ATXN3 expression represents a particularly promising therapeutic strategy. Peptide nucleic acids (PNAs) and mismatched duplex RNAs targeting CAG repeats have demonstrated encouraging selectivity profiles in patient-derived fibroblast models . These approaches exploit structural differences between wild-type and expanded CAG repeats, potentially offering disease-modifying treatments with reduced off-target effects compared to non-selective silencing strategies.